Variant proteins have been produced where a certain amino acid residue of a protein is substituted with an amino acid other than 20 canonical amino acids (referred to as an unnatural amino acid hereinafter) involved in typical protein synthesis. It has been advocated that the protein which contains unnatural amino acids is referred to as the alloprotein (Koide et al., Proc. Natl. Acad. Sci. USA, 85:6237-41, 1988). It makes finer and systematic modification possible that the certain residue can be also replaced by the unnatural amino acid, compared to the cases where the replacements occur among 20 types of natural amino acids. Also, the amino acid with characteristic fluorescent property, the amino acid of which structure can be optically controlled, the amino acid with a reaction group applicable as a optic crosslinker, and the like have been introduced into the proteins.
There are some techniques to produce the alloproteins. Koide et al. made Escherichia coli incorporate an unnatural amino acid added to medium, which was used instead of one certain type of the canonical amino acids to produce the alloprotein. However, only 20 types together with canonical amino acids and unnatural amino acids can be used in this technique (Koide et al., Proc. Natl. Acad. Sci. USA, 85:6237-41, 1988).
Alternatively, the alloprotein is produced by adding the suppressor tRNA which has been aminoacylated beforehand in a separate system to a cell-free translation system (Noren et al., Science, 244:182-8, 1989). The disadvantage of this method includes a necessity to prepare aminoacyl-tRNA in a large amount.
In order to prepare the protein comprising 21 types of amino acids including the unnatural amino acid in the larger amount, it is necessary to construct an artificial genetic code system in which the tRNA attaching the unnatural amino acid is aminoacylated by its cognate aminoacyl-tRNA synthetase (aaRS) in the system where the translation reaction is conducted. The aminoacyl-tRNA synthetase is an enzyme which specifically attaches the amino acid to the tRNA, and 20 types occur corresponding to respective 20 types of canonical amino acids for each biological species excluding some exceptions. In a cell, these enzymes determine the type of the amino acid assigned to the genetic code where basically one type of such an aaRS exists for every amino acid. For instance, tyrosyl-tRNA synthetase (TyrRS) which is one of aminoacyl-tRNA synthetase (aaRS) discriminates tRNA for tyrosine from the other tRNA for the other amino acids, and makes it attach to only tyrosine but not the other amino acids.
In the meanwhile, Wang et al. expressed TyrRS variants derived from Methanococcus janasii modified so as to attach O-methyltyrosine specifically and amber suppressor tRNA engineered by the modification of tyrosine tRNA derived from the same organism, in E. coli (Wang et al., Science, 292:498-500, 2001). This TyrRS and tyrosine tRNA from Methanococcus janasii do not react with tRNA and aaRS from E. coli, respectively. Thus, it has been reported that O-methyltyrosine is incorporated specifically corresponding to an amber codon in this study.
In order to construct such an artificial genetic code system, it is critical that a pair of aaRS and tRNA which does not react with aaRS from the host is found and that the aaRS variant which reacts specifically with the unnatural amino acid is developed. As the aaRS which reacts specifically with the unnatural amino acid, only TyrRS modification specific for O-methyltyrosine mentioned above has been known so far.
As tyrosine analogues substituted at position 3, there are DOPA involved in intercellular signal transduction, 3-iodotyrosine capable of becoming a target site of site-specific labeling in the protein, and the like. These have been known as the unnatural amino acids which exert physiological activity. Therefore, it is desired to obtain TyrRS specific for the tyrosine analogues substituted at position 3. No TyrRS variant has been known so far, which incorporates such tyrosine analogues substituted at position 3 more efficiently than tyrosine. The TyrRS variant was reported at an academic meeting, which incorporates the tyrosine analogues substituted at position 3 more efficiently than the wild-type TyrRS. However, since this variant incorporates tyrosine and the tyrosine analogue at a similar efficiency, it is inappropriate for inserting only the unnatural amino acid in the certain site of the protein.